1. Field of the Invention
The present invention lies in the field of medicine wherein immunomodulators or vaccines are given for modulation, stimulation, and control of the immune response. 2. The Prior Art
What is considered to be the closest prior art consists of liposome-lipophilic muramyl dipeptide analogs and liposome-lymphokines developed for delivery of immunomodulators to macrophages (1-9). A number of papers have described delivery vehicles using intact low density lipoprotein (such as the native unmodified blood, serum or lymphatic lipid particles) as carriers for cytotoxic or therapeutic non-peptide drugs (10-21).
One patent to Pittman, U.S. Pat. No. 4,466,951, Aug. 21, 1984 (22) has suggested using acetyl-LDL to carry covalently bound cellulobiose-therapeutic drug conjugates, however, the chemistry described in this patent for covalently coupling to free amino groups is inappropriate and does not apply to acetyl-LDL since free amino groups have already been acetylated making them unavailable for this purpose.
In contrast to the above, the present invention makes no claims for the covalent coupling of peptide or chemical entities to the acetyl-LDL since that procedure will interfere with the receptor-mediated uptake by macrophages. In both the above Pittman patent (22) and the paper by Nagelkerke, et al., (23) the concept of acetylated-LDL for delivery of immunomodulators, such as lipophilic peptide analogs or vaccines is not contemplated. The blood lipid carrier lipoproteins, such as HDL and LDL chylomicrons have been proposed for vasular site-specific delivery of diagnostic radiopaque agents such as cholesterol iopanoate and polyiodinated 2-substituted triacylglycerols for x-ray organ visualization (24,25,26) with no relation to immune action.
Historically, monoclonal antibodies or other antigens developed for clinical utility have been given systemically as the free antigen or suspended in a non-specific vehicle where they form tissue depots. Alternatively, antigens have been covalently bound or adsorbed to liposomes and synthetic microsphere particles (27-34). The disadvantage of these latter so-called "magic bullet" directed liposomes and microspheres is their large particle size which makes them incapable of penetrating vasular surfaces which restricts their targeted usefulness.
What distinguishes our invention from the prior art is the utilization of the presence of intrinsic cell receptor mediated endocytotic activity as the target for our acetyl-LDL carrier. The acetylated LDL carries lipophilic peptide analogs acting as immunomodulators or vaccines to macrophage processing or reactive sites. The peptides to be carried by acetyl-LDL are modified chemically when necessary to make or enhance their lipid solubility with lipophilic functional groups to render them of sufficient hydrophobicity to partition and anchor in the lipophilic domain of the acetyl-LDL or are already lipophilic. As mentioned previously, this entry of the immunomodulation or vaccine into the acetyl-LDL relates to lipid solubility and is not a direct covalent, coupling to free amino groups on the acetyl-LDL. Other advantages of the acetyl-LDL carrier are its small size (.about.20 nm), native source and its more natural metabolism that enhances its host compatibility and therapeutic index.
In regard to the action of acetyl-LDL carriers as vaccines or immunoadjuvants previous work has shown that the class II products of the major histocompatibility complex (MHC) involved in immune response are molecules which are cell-surface receptors that bind peptide fragments (of degraded or `processed` antigens) of foreign and self proteins and present those bound peptides to T lymphocytes (35,36). These class II molecules are capable of binding a large number of peptides of different sequence and are thus general, relatively nonspecific, peptide receptors. The ability to present such peptides to T lymphocytes is a widespread phenomenon depending on macrophages and B cells which express these class II MHC products.
Several groups have now been able to identify those portions of a protein which actually represent the immunogenic portion of the molecules and have shown that these peptide segments are capable of being recognized by the T lymphocytes which generate an immune response (35-38). Morever, it is possible to chemically synthesize such peptides and through the addition of chemical groups or additional amino groups alter the chemical properties of such peptides without affecting their recognition by the immune system (39).
Several factors affect the induction of an immune response by protein vaccines: First, the site of administration (parenterally or orally), the amount of protein administered, and the delivery vehicle (alum or physiologic buffered saline [PBS]. The immunization of humans has required the injection of many low doses of antigen given over a number of years to ensure an efficacious immunization. This latter point may reflect the fact that many self (endogenous) proteins are also being processed which require the immunized peptide to complete for binding to the class II MHC molecule (36,37). The successful use of adjuvants in animal models allows less peptide from having to be injected into the animal. However, such approaches have been limited in human immunizations because of the associated side effects. The application of chemically synthesized peptides to serve as immunogenic compounds currently requires production in large quantities because of the amounts required to effectively immunize clinically.
The use of acetyl-LDL provides a clinical solution to the requirement for large amounts of peptide for the immunization of individuals. The ability to render any immunogenic peptide lipophilic by the addition of hydrophobic residues provides a method for partitioning the peptide into the acetyl-LDL particle. Additionally, many of the peptides which have been studied are actually lipophilic in nature since the areas of a native globular protein which appear to be recognized by the T lymphocytes are amphoteric stretches of 10-15 amino acids which go from hydrophilic to hydrophobic in nature (35,38). Thus, a lipophilic peptide can enter and be partitioned within the acetyl-LDL particle. Because of the properties of this acetyl-LDL lipoprotein particle to selectively bind to macrophages and its inherent adjuvant action, it provides an efficacious carrier of immunogenic peptides. This approach requires only small amounts of peptides and may avoid the need to immunize individuals for more than a single dose.
As discussed, the advantages of acetyl-LDL in our invention are its roles in carrying antigen or macrophage stimulating agents to selectively responding cells of the immune system to enhance their action.
In regard to macrophage action, there is a mutual stimulation between macrophages and immunomodulators such as seen with interferon which produces macrophage stimulation which results in further interferon induction with associated anti-tumor, anti-viral action (40).
A list of references are set out further herein and incorporated in this application for providing a very specific description of all of the prior art known to the inventors.